The ability of cells to create and tightly regulate the chemical environment in specialized compartments is critical to many fundamental biological processes. Lysosomes and the extracellular compartment (ECC) of bone resorbing osteoclasts must maintain an acidic interior pH, around 4.5, to effectively destroy macromolecules and bone matrix, respectively. These cellular compartments are intimately linked, since the ECC is derived from lysosomes; and they both use a common set of membrane proteins to achieve a low lumenal pH. Dysfunction in the acidification process in lysosomes can lead to lysosomal storage diseases; and aberrant bone resorption by osteoclasts is associated with a number of bone diseases including osteoporosis, which affects 10 million people in the United States and causes significant morbidity and mortality. Despite the profound influence that the proper functioning of osteoclasts and lysosomes has on human health, there is no unified understanding of how pH is regulated in these systems. In recent years, there has been a dramatic increase in our knowledge of the proteins involved in acidification of lysosomes and ECC and their biophysical properties. Several critical proteins in this process include the V-ATPase proton pump, ClC-7 chloride antiporter, and the proton leak channel. The goal of this proposal is to create comprehensive mathematical models of pH regulation in lysosomes (Aim 1) and the ECC of osteoclasts (Aim 2). We will start by individually calibrating the ionic flux of each relevant transporter and ion channel over a large range of environmental conditions. These performance surfaces will then be used as inputs in the construction of ordinary differential equation (ODE) based models of ion regulation in lysosomes and the ECC. The ODE models will be calibrated against experimental data. We will also create 3D models of both cellular compartments, and carry out acidification studies using Monte Carlo simulations. We expect that our studies will help resolve a controversy in the field regarding the identity of the counter-ion flux required for proton pumping, and the models will guide new experiments. Additionally, our models will reveal potential drug targets for inhibiting ECC acidification that could lead to new osteoporosis treatments. PUBLIC HEALTH RELEVANCE: This study, in part, is to determine how osteoclasts acidify the extracellular compartment to degrade bone. Our results will impact our understanding of osteoclast dysfunction, which is the cause of most adult skeletal diseases. Our studies will also help identify new drug targets for the treatment of osteoporosis. 1